Germinal Cells Research Articles

Abstract A21: Novel autochthonous mouse models as preclinical tools in the study of MCD/C5 DLBCL

Abstract Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically very heterogeneous disease. Several [GK1] [RW2] recent studies attempted to give structure to the genetic heterogeneity by clustering cases based on their mutational profiles. Clusters MCD by Schmitz et al. and C5 by Chapuy et al. are amongst the clusters enriched for cases with particularly inferior prognosis. Both clusters are defined by mutations in MYD88 and CD79B as well as high levels of BCL2 expression and recurring mutations in transcriptional regulators of the plasmacytic differentiation process (PRDM1, SPIB). We have generated genetically-engineered mouse models that mimic key alterations of MCD/C5 DLBCL: Hallmark mutations in MYD88 and CD79B, loss of PRDM1 and overexpression of BCL2/SPIB. We could show that those mice develop clonal lesions, which morphologically resemble DLBCL. In the presence of a lesion that prevents plasmacytic differentiation (overexpression of Spib, loss of Prdm1), the forming lesions display signs of an earlier stage of B cell development, specifically histologic and transcriptomic similarities to germinal center cells. In contrast, allelic combinations without differentiation block lead to lymphomas that express CD138 and lack B220 expression, suggesting a more plasmacytic/plasmablastic differentiation status. The presence or absence of a Cd79b p.Y195H mutation has no notable effect on the immunophenotype of the lymphoma. However, its presence correlates with enrichment of BCR signaling gene set expression in lymphoma tissue and significantly increased sensitivity to inhibition of Bruton’s Tyrosine Kinase (BTK) by ibrutinib in our model systems. Taken together, we have established a set of murine models which mimic different genetic features of C5/MCD DLBCL. We have extensively characterized these models and show their usability as preclinical tools for therapeutic studies. Citation Format: Ruth Fluemann, Julia Hansen, Benedikt Pelzer, Tim Lohmann, Ilmars Kisis, Reinhard Büttner, Thorsten Persighel, Nima Abedpour, Martin Peifer, Ron Daniel Jachimowicz, Hans Christian Reinhardt, Gero Knittel. Novel autochthonous mouse models as preclinical tools in the study of MCD/C5 DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A21.

Dual inhibition of the Echinococcus multilocularis energy metabolism.

Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite Echinococcus multilocularis. Current chemotherapeutic treatment options rely on benzimidazoles, which have limited curative capabilities and can cause severe side effects. Thus, novel treatment options are urgently needed. In search for novel targetable pathways we focused on the mitochondrial energy metabolism of E. multilocularis. The parasite relies hereby on two pathways: The classical oxidative phosphorylation including the electron transfer chain (ETC), and the anaerobic malate dismutation (MD). We screened 13 endochin-like quinolones (ELQs) in vitro for their activities against two isolates of E. multilocularis metacestodes and isolated germinal layer cells by the phosphoglucose isomerase (PGI) assay and the CellTiter Glo assay. For the five most active ELQs (ELQ-121, ELQ-136, ELQ-271, ELQ-400, and ELQ-437), EC50 values against metacestodes were assessed by PGI assay, and IC50 values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome b, was analyzed. This allowed for a limited structure activity relationship study of ELQs against E. multilocularis, including analyses of the inhibition of the two functional sites of the cytochrome b. By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the E. multilocularis cytochrome bc1 complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against E. multilocularis metacestodes. These results were confirmed by transmission electron microscopy. ELQ-400 treatment increased levels of parasite-released succinate, the final electron acceptor of the MD. This suggests that the parasite switched to MD for energy generation. Therefore, MD was inhibited with quinazoline, which did not induce damage to metacestodes under anaerobic conditions. However, it reduced the production of succinate compared to control treated parasites (i.e., inhibited the MD). The combination treatment with quinazoline strongly improved the activity of the bc1 inhibitor ELQ-400 against E. multilocularis metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of E. multilocularis is a possible novel treatment approach for alveolar echinococcosis, and possibly also other foodborne diseases inflicted by platyhelminths, which cause substantial economic losses in livestock industry.

EmCyclinD-EmCDK4/6 complex is involved in the host EGF-mediated proliferation of Echinococcus multilocularis germinative cells via the EGFR-ERK pathway

The larval stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. The tumor-like growth and development of the metacestode larvae within host organs are driven by a population of somatic stem cells, the germinative cells, which represent the only proliferative cells in the parasite. Host-derived factors have been shown to promote germinative cell proliferation. Since cells sense the external signal mainly in G1 phase of the cell cycle, host factors are expected to exert impacts on the machinery regulating G1/S phase of the germinative cells, which still remains largely unknown in E. multilocularis. In this study, we described the characterization of two key members of the G1/S phase cell-cycle regulation, EmCyclinD and EmCDK4/6. Our data show that EmCyclinD and EmCDK4/6 display significant sequence similarity to their respective mammalian homologs, and that EmCyclinD interacts with EmCDK4/6, forming a kinase-active complex to activate its substrate Rb1. EmCyclinD was actively expressed in the germinative cells. Addition of human EGF caused an elevated expression of EmCyclinD while inhibition of the EGFR-ERK signaling pathway in the parasite reduced the expression of EmCyclinD and downstream transcriptional factors. Treatment with Palbociclib, a specific CDK4/6 inhibitor, downregulated the expression of cell cycle-related factors and impeded germinative cell proliferation and vesicle formation from protoscoleces. Our data demonstrated that the EmCyclinD-EmCDK4/6 complex participates in the cell cycle regulation of germinative cells which is mediated by host EGF via the EGFR-ERK-EmCyclinD pathway in E. multilocularis.

Cell repertoire and proliferation of germinative cells of the model cestode Mesocestoides corti.

The phylum Platyhelminthes shares a unique population of undifferentiated cells responsible for the proliferation capacity needed for cell renewal, growth, tissue repair and regeneration. These cells have been extensively studied in free-living flatworms, whereas in cestodes the presence of a set of undifferentiated cells, known as germinative cells, has been demonstrated in classical morphology studies, but poorly characterized with molecular biology approaches. Furthermore, several genes have been identified as neoblast markers in free-living flatworms that deserve study in cestode models. Here, different cell types of the model cestode Mesocestoides corti were characterized, identifying differentiated and germinative cells. Muscle cells, tegumental cells, calcareous corpuscle precursor cells and excretory system cells were identified, all of which are non-proliferative, differentiated cell types. Besides those, germinative cells were identified as a population of small cells with proliferative capacity in vivo. Primary cell culture experiments in Dulbecco's Modified Eagle Medium (DMEM), Echinococcus hydatid fluid and hepatocyte conditioned media in non-reductive or reductive conditions confirmed that the germinative cells were the only ones with proliferative capacity. Since several genes have been identified as markers of undifferentiated neoblast cells in free-living flatworms, the expression of pumilio and pL10 genes was analysed by qPCR and in situ hybridization, showing that the expression of these genes was stronger in germinative cells but not restricted to this cell type. This study provides the first tools to analyse and further characterise undifferentiated cells in a model cestode.

Morphometrical and Histological Analyses of the Epithelial Lining of Male Reproductive System in Wistar Rats Following Administration of Neem Leaves Extract

Background: Neem leaves (Azadirachta indica L.) have been used for many therapeutic purposes and medicinal applications. The extract of this plant has been used in both male and female genders as a traditional agent to prevent early pregnancy. In this study, the effect of this extract was investigated histologically and morphometrically on the germinal epithelia of the seminiferous tubules, epididymis and secretory epithelia of rats’ prostate glands. Methods: Twenty male albino rats were divided into four groups of five each and administered the extract at a concentration of zero, 100, 200 or 400 mg/kg of the body weight for 50 consecutive days. These rats were sacrificed and the male reproductive organs were removed, weighed and processed for routine histological examinations. The micrographs were analyzed and the structural changes in the epithelial lining and morphometric analyses were recorded, which included measuring the epithelial thickness in the seminiferous tubules, epididymis and secretory prostatic epithelia. Results: The extract was found to reduce the rats’ weight; decreased both the weight and dimension of the testes; reduced the number of germinal epithelial lining cells in the seminiferous tubules of the testes, the epididymal and prostatic secretory epithelial cells. Conclusion: The histological alterations were most significant in response to the treatment with the extract at 200 mg/kg of the rats with the greatest damages observed in the epithelial lining. The deleterious effects of the extract were found to be dose-dependent and this corroborates the use of this extract as a contraceptive in animal models, and potentially in humans.

Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster

The nascent polypeptide-associated complex (NAC) consisting of α- and β-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. Here we report on discovering the germline-specific NACαβ paralogs (gNACs), whose β-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single αNAC gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and β subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-β subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. We revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-β sequences of Drosophila species are positionally conserved despite these disordered regions are drastically different. We propose the IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells. Our findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and β-subunits based on assessing their depletion effects on the fly viability and gonad development.

P-073 SARS-CoV2 infection in human testis and sperm: in vivo and in vitro studies

Study questionCan severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) enter somatic and germinal cells of human testis or ejaculated sperm, thus affecting male reproductive function?Summary answerThis research provides a biological background of the potential route for infection of SARS-CoV-2 and may enable rapid deciphering of COVID-19-induced male-related reproductive disorders.What is known alreadyEpidemiologic studies suggest a significant male sex susceptibility for severe COVID19 symptoms. SARS-CoV-2 is known to affect certain cell types based on their expression of angiotensin-converting enzyme 2 (ACE2) and Transmembrane serine protease2 (TMPRSS2). ACE2 makes available the binding site for the Spike protein of SARS-CoV-2. TMPRSS2 facilitates virus entry by cleaving the S antigen into S1 (the active binding site). Several studies reported the presence of ACE2 in Leydig and Sertoli cells as well as in germ cells from spermatogonia to spermatozoa. These data suggest that the human testis and gametes are a target for SARS-CoV-2.Study design, size, durationTo address this question, we examined the gene expression profile of SARS-CoV-2-associated receptors and proteases (ACE2-TMPRSS2) as well as their protein expression and localization in testicular tissue of males undergoing diagnostic surgery and in sperm of healthy, normozoospermic donors referring to the Unit of Assisted Reproduction, Siena University Hospital, from April 2020 to January 2022.Participants/materials, setting, methodsAssays were performed on tissue biopsies (n = 3) or on freshly ejaculated sperm of men (n = 6) undergoing routine semen analysis after granting informed consent. To this end, tissue biopsies and ejaculated sperm have been co-cultivated for 5-12h with infected VERO E6 cells. Immunoelectron microscopy, in situ hybridization, and highly sensitive digital droplet PCR analysis, have been used to assess if particles containing SARS-CoV- 2 antigens can be detected in testicular tissue or spermatozoa in vitro.Main results and the role of chanceWe carefully investigated the presence of the SARS-CoV-2 virus and the possible effect on male fertility, both at the molecular and ultrastructural levels. To elucidate the mechanisms underlying virus infection in germ cells and male gametes, we analyzed the expression of both ACE2 and TMPRSS2. Both genes are expressed in testes and ejaculated sperm, confirming the possibility that SARS-CoV2 can enter these cells. We also provided evidence of the expression of the corresponding proteins in both testis and sperm by immunofluorescence assays. The immunofluorescence staining of paraffin-embedded slices of testicular tissue with anti-ACE2 antibody revealed a strong signal in Leydig cells. However, ACE2 staining was also present in human Sertoli cells and was concentrated in the adluminal half of the cell, i.e. surrounding spermatocytes and spermatids.The analysis of TRPMSS2 showed similar results. Indeed, this protease is localized mainly in the interstitium, at the level of Leydig cells. This is an intriguing datum, since, while Leydig cells may be considered as a high-risk cells because of the co-expression of TMPRSS2 and ACE2, germ cells may not be at increased risk of ACE2and TMPRSS2-mediated viral entry and spread, given the lack of co-expression in these testicular cell type.Limitations, reasons for cautionThe low number of analyzed samples may limit the statistical power of this study. Whether the SARS-CoV-2 infection change the gene expression profile of other SARS-CoV-2-associated proteases is under investigationWider implications of the findingsThis validated RT-PCR assay allows reliable screening of SARS-CoV-2 in sperm, useful for investigating the presence of the virus in patients undergoing ART, as well as for explaining the molecular and cellular mechanisms related to the gender specificity of the more severe infection-related symptoms.Trial registration numberNot applicable

Effects of N-Acetylcysteine Supplementation on Oxidative Stress and Expression of Apoptosis-Related Genes in Testicular Tissue of Rats Exposed to Lead.

Lead occupational exposure is now a main concern in the modern world. Lead is a non-biodegradable element with multi-devastating effects on different organs. Acute or chronic exposure to lead is reported to be one of the most important causes of infertility both in males and females basically by inducing oxidative stress and apoptosis. The current study scrutinized the mitigating effects of N-acetylcysteine (NAC) on lead toxicity, oxidative stress, and apoptotic/anti-apoptotic genes in the testis tissues of male rats. Rats were randomly divided into a control group (G1) and four study groups treated with single and continuous doses of lead with and without NAC administration. Malondialdehyde (MDA), total antioxidant capacity (TAC), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were analyzed as oxidative stress biomarkers and the expression of apoptosis-related genes was studied using RT-PCR. Continuous exposure to lead caused a significant decrease in sperm count, motility, viability, and morphology (P < 0.001). Number of germinal cells, Leydig cells, spermatocytes, and the diameter of seminiferous tubule were significantly decreased (P < 0.001) in G3 group. Continuous exposure to lead significantly decreased TAC content, but increased the levels of MDA and 8-OHdG (P < 0.001). Administration of continuous dose of lead dramatically increased expression of Bax, Caspase-3, Caspase-8, Cytochrome-C, MMP2, and MMP9 genes in testicular tissue. NAC treatments not only improved morphological changes and sperm quality, but also enhanced antioxidant balance and modulated apoptosis process in testicular tissue of rats. Lead exposure strongly motivated testicular cells towards apoptosis, caused an oxidant/antioxidant imbalance, and decreased sperm quality along with morphological changes in testis cells. NAC treatments was associated with protective effects on testicular tissue mainly by rebalancing of the antioxidants capacity, as well as downregulation of apoptosis-related genes.

Testicular Assessment of (Okra) Abelmoschus Esculentus Treated Wistar Rats

Results of the histology of the control group showed a normal histology of the seminiferous tubules (round cells), low dose showed seminiferous tubules with acute loss of Germinal cell area (G) and high dose showed testicular regression (loss of interstitial space, IT), tubular architecture and Germ layer (G).

Plasmablastic lymphoma with MYC::IGH fusion and BCL2 rearrangement.

A 50-year-old male with history of nodal stage III diffuse large B-cell lymphoma (DLBCL), refractory to R-CHOP, presented with vomiting, abdominal pain, and an umbilical mass. Abdominal computed tomography (CT) scan showed bulky retroperitoneal adenopathy and possible bowel obstruction. Histologic examination of the umbilical mass needle biopsy demonstrated diffuse atypical cells with medium to large nuclei, prominent central nucleoli, and eccentrically placed amphophilic cytoplasm (Figure 1, top left panel, objective 100×). The original lymphoma was of germinal center B-cell phenotype, was MYC-negative by immunohistochemistry (IHC), and had BCL2 rearrangement (BCL2-R) without MYC-R by interphase fluorescence in situ hybridization (FISH). In contrast, the umbilical mass neoplastic cells expressed plasma cell markers CD138 (Figure 1, top right, objective 60×) and MUM1 (Figure 1. Bottom left, objective 60×) and MYC (90%), were lambda light chain restricted, had a very high Ki-67 proliferation index (>90%), and were negative for all B-cell markers, germinal center cell markers, and Epstein-Barr virus. Interphase FISH studies identified BCL2-R and IGH::MYC fusion (Figure 1, bottom right, Ch 8 centromere: aqua probe, MYC [8q24]: red probe, IGH [14q32]: green probe; yellow fusion indicates IGH::MYC) without BCL6-R. These findings are characteristic of plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. This likely represents transformation of the patient's prior DLBCL, with clonal evolution characterized by acquisition of MYC-R. Transformation of follicular lymphoma to plasmablastic lymphoma with MYC-R and BCL2-R has been previously described, but to our knowledge this is the first reported case of transformation of DLBCL to plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. The “double-hit” cytogenetic features may portend an aggressive clinical course, similar to what has been observed in “high-grade B-cell lymphomas with MYC-R and BCL2-R and/or BCL6-R” (so-called double-hit lymphoma). The authors would like to thank the Mayo Clinic Genomics Laboratory for their continuous assistance and support of our service. The authors declare they have no conflict of interest. The authors received no specific funding for this work. Mohammad Barouqa wrote the manuscript. Patricia Greipp reviewed the article and cytogenetic probes. Rebecca King reviewed the article and stains. Ellen D. McPhail supervised the manuscript and finalized it.

Effects of flumethrin on the germinative cells of engorged females of Rhipicephalus annulatus

Chemical acaricides are widely used for the effective control of ticks in India. The synthetic pyrethroids, are one of the most popular chemical acaricides with selective neurotoxic potential. Flumethrin is a type II synthetic pyrethroid used extensively in veterinary practice in India. The present study was undertaken to evaluate the cytotoxic effects of flumethrin on the engorged females of Rhipicephalus annulatus using entomological parameters, histology, electron microscopy and relative quantification of receptors of dopamine and GABAB mRNAs. Adult immersion test (AIT) using flumethrin (100 ppm), revealed twenty per cent mortality of ticks, hundred per cent inhibition of fecundity and complete blocking of hatching of the laid eggs. Microscopic analysis of the structure of the ovaries after 24 h of treatment with flumethrin (90 ppm) revealed changes, viz., reduction in size with the presence of amorphous material inside stage I oocytes, wrinkled boundary and chromatin fragmentation of nucleus of stage II oocytes, vacuoles around the germinal vesicle, thickening of the nuclear membrane and chromatin clumping of stage III oocytes and reduction in size and shape of mature stage IV and V oocytes. Also, a large number of vacuoles were observed throughout the pedicel cell region of stage II and III oocytes. Ultrastructurally, irregular nuclear membrane, swelling as well as crystolysis of mitochondria and detachment of external and internal layers of the basal lamina of oocytes were the major structural alterations confirming direct damaging effects of flumethrin on the germinative cells. The relative quantification of the expression of dopamine D1, dopamine D2 and GABAB receptors by quantitative real-time PCR (qRT PCR), revealed the upregulation of dopamine D1 receptor and downregulation of receptors of dopamine D2 and GABAB in the ovary of treated ticks.

Identification of CD4+ T cells with T follicular helper cell characteristics in the pig

T follicular helper (Tfh) cells provide help to germinal center B cells for affinity maturation, class switch and memory formation. Despite these important functions, this subset has not been studied in detail in pigs due to a lack of species-specific antibodies. We investigated putative Tfh cells from lymphoid tissues and blood of healthy pigs by using cross-reactive antibodies for inducible T-cell costimulator (ICOS) and B-cell lymphoma 6 (Bcl-6). In lymph nodes, we identified a CD4+ T cell population with an ICOS+Bcl-6+CD8α+ phenotype, reminiscent of human and murine germinal center Tfh cells. Within blood-derived CD4+ T cells, sorted ICOShiCD25- and ICOSdimCD25dim cells were able to induce the differentiation of CD21+IgM+ B cells into Ig-secreting plasmablasts. Compared to naïve CD4+ T cells, these two phenotypes were 3- to 7-fold enriched for cells expressing the Tfh-related transcripts CD28, CD40LG, IL6R and MAF, as identified by single-cell RNA sequencing. These results provide a first characterization of Tfh cells in swine and confirm their ability to provide B-cell help.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The alterations of blood-testis barrier in experimental testicular ınjury models

The blood-testis barrier is found between the Sertoli cells and divides the seminiferous tubule epithelium into basal and adluminal compartments. The germinal cell renewal, differentiation and cell cycle progression up to the preleptotene spermatocytes stage take place in the basal compartment, however, meiosis, spermiogenesis and spermiation take place in the adluminal compertment. The blood-testis barrier consists of tight junctions as well as ectoplasmic specialisations, desmosomes and gap junctions to create specific microenvironment for the completion of spermatogenesis to form spermatozoa. The blood-testis barrier is not a static ultrastructure, it undergoes extensive restructuring during the seminiferous tubule epithelial cycle of spermatogenesis to allow the transit of preleptotene spermotocytes at the blood-testis barrier from basal compartment towards the adluminal compartment. The functions of the blood-testis barrier include preventing the transport of biomolecules into the paracellular space, forming an immunological barrier, separating cellular processes during the spermatogenic epithelial cycle, and establishing the cellular polarity of the seminiferous tubule. However, various environmental conditions, chemotherapeutic agents, toxic substances and lifestyle have degenerative effects on blood-testis barrier, resulting in testicular damage, altered sperm parameters and ultimately male infertility. The alterations in morphological and molecular organization of blood-testis barrier in different experimentally induced testis injury models are reviewed in this article.

A Random Forest-Based Genome-Wide Scan Reveals Fertility-Related Candidate Genes and Potential Inter-Chromosomal Epistatic Regions Associated With Age at First Calving in Nellore Cattle.

This study aimed to perform a genome-wide association analysis (GWAS) using the Random Forest (RF) approach for scanning candidate genes for age at first calving (AFC) in Nellore cattle. Additionally, potential epistatic effects were investigated using linear mixed models with pairwise interactions between all markers with high importance scores within the tree ensemble non-linear structure. Data from Nellore cattle were used, including records of animals born between 1984 and 2015 and raised in commercial herds located in different regions of Brazil. The estimated breeding values (EBV) were computed and used as the response variable in the genomic analyses. After quality control, the remaining number of animals and SNPs considered were 3,174 and 360,130, respectively. Five independent RF analyses were carried out, considering different initialization seeds. The importance score of each SNP was averaged across the independent RF analyses to rank the markers according to their predictive relevance. A total of 117 SNPs associated with AFC were identified, which spanned 10 autosomes (2, 3, 5, 10, 11, 17, 18, 21, 24, and 25). In total, 23 non-overlapping genomic regions embedded 262 candidate genes for AFC. Enrichment analysis and previous evidence in the literature revealed that many candidate genes annotated close to the lead SNPs have key roles in fertility, including embryo pre-implantation and development, embryonic viability, male germinal cell maturation, and pheromone recognition. Furthermore, some genomic regions previously associated with fertility and growth traits in Nellore cattle were also detected in the present study, reinforcing the effectiveness of RF for pre-screening candidate regions associated with complex traits. Complementary analyses revealed that many SNPs top-ranked in the RF-based GWAS did not present a strong marginal linear effect but are potentially involved in epistatic hotspots between genomic regions in different autosomes, remarkably in the BTAs 3, 5, 11, and 21. The reported results are expected to enhance the understanding of genetic mechanisms involved in the biological regulation of AFC in this cattle breed.

SHORT-TERM EFFECT OF Naja naja SHED SKIN EXTRACT ON HISTOPATHOLOGY OF TESTIS, EPIDIDYMIS AND VAS DEFERENS OF SWISS ALBINO MICE

Ancient literature reveals that snake shed skin had been frequently used to treat several diseases like glaucoma, hernia, psoriasis, hemorrhoids, etc. Previously, it was reported that Naja naja shed skin extract causes temporary cessation of the estrous cycle and altered female reproductive hormones in rodents, and the probable bioactive molecule behind such alterations was also identified. Based on this information, the present study was designed to establish the effect of Naja naja shed skin extract on the male reproductive system of Swiss albino mice. The extract was prepared with physiological saline and was injected intraperitoneally in the male mice for 7 consecutive days @ 10 mg kg-1 body weight. The short-term histopathological studies were performed by Haematoxylin and Eosin staining and the sperm morphology was analyzed by using bright-field microscopy. The snake skin extract markedly changed the gross histological architecture of the seminiferous tubules including disorganization of germinal epithelial cells, accumulation of exfoliated cells in the lumen, tailless sperm in the epididymis, disorganized basal cells in vas deferens as well as marked alteration of sperm morphology. The present work with snake shed skin strongly indicates that it is not simply a biological waste, but it might be a treasure house of many bioactive compounds.

SEX REVERSAL IN ANIMAL KINGDOM : SPECIAL EMPHASIS ON ROLE OF STEROID HORMONES

Sexual fate is no longer seen as an irreversible deterministic switch set during early embryonic development but as an ongoing battle for primacy between male and female developmental trajectories. That sexual fate is not final and must be actively maintained via continuous suppression of the opposing sexual network creates the potential for flexibility into adulthood. In many Animals, sexuality is not only extremely plastic, but sex change is a usual and adaptive part of the life cycle. Sequential hermaphrodites begin life as one sex, changing sometime later to the other, and include species capable of protandrous (male- to-female), protogynous (female-to-male), or serial (bidirectional) sex change. Natural sex change involves coordinated transformations across multiple biological systems, including behavioural, anatomical, neuroendocrine, and molecular axes. This can be caused by human pollutants, including herbicides, which can act as estrogen promoters or inhibitors, which would respectively increase or decrease the number of female offspring, through controlling aromatase. This review project paper highlights the biological processes underlying this amazing transformation, Gonadal restructuring among germinal and somatic cell interaction, Neuroendocrine control of sex change and Stress response and role of Steroid hormones-balance between 17beta-estradiol &amp; 11- ketotestosterone in sex changes, Endocrine disruption on Changing of Sexual Behaviour, which remains poorly understood, but where new genomic technologies are significantly advancing our understanding of how sex change is initiated and progressed at Hormonal and molecular levels. Knowledge of how a usually committed developmental process remains plastic in sequentially hermaphroditic fishes, Amphibians and Birds are relevant to understanding the evolution and functioning of sexual developmental systems in vertebrates generally, as well as pathologies of sexual development in mammals, some time observed in nematodes and Gastropodes also. Keywords- sex change, hormones, hermaphrodism

Dietary Zinc Deficiency Increases Bisphenol A Toxicity in Diabetic Rat: Studies on the Testicular and Epididymal Pathophysiology

Bisphenol A (BPA), an endocrine disrupter extensively used in plastics products, that causes male reproductive dysfunction in humans and experimental systems. Type 1 diabetes (T1D) associates hyperglycaemia dysregulate the spermatogenesis and antioxidant status in the testes, which negatively impact on male fertility in adults. Dietary zinc (Zn) deficiency is a global health concern, because majority of the individuals are suffering from certain range of Zn‐deficiency. Zn plays an important role in the regulation and maintenance of testicular physiology and the overall reproductive health. The present study was aimed to investigate the influence of Zn‐deficiency on BPA induced germinal cell and male gonadal toxicity in diabetic rat. Sprague Dawley rats (4 weeks old) were randomly divided into eight different groups; like control (standard pellet diet and aquapure drinking water), BPA (10 mg/kg/day), Zn‐deficient diet (ZDD; Zn‐deficient diet), diabetic (DIA), BPA+ZDD, BPA+DIA, ZDD+DIA and BPA+ZDD+DIA for four weeks. Diabetes was induced by injecting single dose of streptozotocin (55 mg/kg) freshly prepared in cold citrate buffer at pH 4.5. The evaluation of various end‐points such as assessment of body and organ weights, biochemical estimations, sperm parameters. testes and epididymis histopathology, quantification of testicular DNA damage and sperm apoptosis were conducted. Testicular 8‐OHdG immunoexpression as well as catalase and OCT4 expressions were carried out by western blot analysis. The results showed that dietary Zn‐deficient condition significantly increased the BPA toxicity by decreasing body and organ weight, serum albumin and increasing glucose, SGPT, SGOT and BUN significantly in diabetic condition. Further, significant decrease in sperm count, sperm motility and increase in sperm deformities were observed. The Zn‐deficiency significantly increased the BPA induced testicular and epididymal tissue injury, elevated testicular 8‐OHdG immunoexpression and sperm apoptosis in diabetic rat. Moreover, BPA exposure significantly decreased the testicular catalase and OCT4 expressions in dietary Zn‐deficient diabetic rat. The present results concluded that dietary Zn‐deficiency exacerbated the BPA induced gonadal toxicity by hypogonadism, increased sperm abnormalities, testicular and epididymal cellular damage, testicular DNA damages, sperm apoptosis as well as decreased the testicular catalase and OCT4 expressions in diabetic rat.

Lymph node CXCR5+ NK cells associate with control of chronic SHIV infection.

The persistence of virally infected cells as reservoirs despite effective antiretroviral therapy is a major barrier to an HIV/SIV cure. These reservoirs are predominately contained within cells present in the B cell follicles (BCFs) of secondary lymphoid tissues, a site that is characteristically difficult for most cytolytic antiviral effector cells to penetrate. Here, we identified a population of NK cells in macaque lymph nodes that expressed BCF-homing receptor CXCR5 and accumulated within BCFs during chronic SHIV infection. These CXCR5+ follicular NK cells exhibited an activated phenotype coupled with heightened effector functions and a unique transcriptome characterized by elevated expression of cytolytic mediators (e.g., perforin and granzymes, LAMP-1). CXCR5+ NK cells exhibited high expression of FcγRIIa and FcγRIIIa, suggesting a potential for elevated antibody-dependent effector functionality. Consistently, accumulation of CXCR5+ NK cells showed a strong inverse association with plasma viral load and the frequency of germinal center follicular Th cells that comprise a significant fraction of the viral reservoir. Moreover, CXCR5+ NK cells showed increased expression of transcripts associated with IL-12 and IL-15 signaling compared with the CXCR5– subset. Indeed, in vitro treatment with IL-12 and IL-15 enhanced the proliferation of CXCR5+ granzyme B+ NK cells. Our findings suggest that follicular homing NK cells might be important in immune control of chronic SHIV infection, and this may have important implications for HIV cure strategies.

Expression of the histone lysine methyltransferases SETD1B, SETDB1, SETD2, and CFP1 exhibits significant changes in the oocytes and granulosa cells of aged mouse ovaries.

Histone methylation is one of the main epigenetic mechanisms by which methyl groups are dynamically added to the lysine and arginine residues of histone tails in nucleosomes. This process is catalyzed by specific histone methyltransferase enzymes. Methylation of these residues promotes gene expression regulation through chromatin remodeling. Functional analysis and knockout studies have revealed that the histone lysine methyltransferases SETD1B, SETDB1, SETD2, and CFP1 play key roles in establishing the methylation marks required for proper oocyte maturation and follicle development. As oocyte quality and follicle numbers progressively decrease with advancing maternal age, investigating their expression patterns in the ovaries at different reproductive periods may elucidate the fertility loss occurring during ovarian aging. The aim of our study was to determine the spatiotemporal distributions and relative expression levels of the Setd1b, Setdb1, Setd2, and Cxxc1 (encoding the CFP1 protein) genes in the postnatal mouse ovaries from prepuberty to late aged periods. For this purpose, five groups based on their reproductive periods and histological structures were created: prepuberty (3 weeks old; n = 6), puberty (7 weeks old; n = 7), postpuberty (18 weeks old; n = 7), early aged (52 weeks old; n = 7), and late aged (60 weeks old; n = 7). We found that Setd1b, Setdb1, Setd2, and Cxxc1 mRNAlevels showed significant changes among postnatal ovary groups (P < 0.05). Furthermore, SETD1B, SETDB1, SETD2, and CFP1 proteins exhibited different subcellular localizations in the ovarian cells, including oocytes, granulosa cells, stromal and germinal epithelial cells. In general, their levels in the follicles, oocytes, and granulosa cells as well as in the germinal epithelial and stromal cells significantly decreased in the aged groups when compared the other groups (P < 0.05). These decreases were concordant with the reduced numbers of the follicles at different stages and the luteal structures in the aged groups (P < 0.05). In conclusion, these findings suggest that altered expression of the histone methyltransferase genes in theovarian cells may be associated with female fertility loss in advancing maternal age.